A key intermediate in the preparation of sugar analogues used in the synthesis of nucleosides and vitamins is 2-C-methyl-D-ribono-lactone. As early as 1880, Scheibler described a process for preparing the lactone (John Sowden, “The Saccharinic Acids” in Adv. Carbohydrate Chem. 12:43-46 (1957), citing C. Scheibler, Berichte 13:2212 (1880)). Unfortunately, product yields were only approximately 10% (Id.). At about the same time, H. Kiliani synthesized 2-methyl-D-ribonolactone by treating D-fructose with calcium hydroxide (H. Kiliani, Berichte, 15:2953 (1882), as cited in F. J. Lopez-Herrera et al., J. Carbohydrate Chemistry, 13(5):767-775 (1994)). However, the process required months to run to completion and product yield was only 10% (d. at 768). Kiliani's process, however, enabled him to establish the positions of important functional groups on the compound (John Sowden, “The Saccharinic Acids” in Adv. Carbohydrate Chem. 12:43-46 (1957), citing H. Kiliani, Ann., 213:361 (1883)).
In the early 1960s, Whistler and BeMiller attempted to improve upon Kiliani's synthesis (Roy L. Whistler and J. N. BeMiller, “α-D-Glucosaccharino-1,4-lactone” in Methods in Carbohydrate Chemistry, 2:484-485 (1963)). Whistler and BeMiller added boiling water and calcium hydroxide to D-fructose, flushed the system with nitrogen gas, and repeated the same process. After 2 weeks; the mixture then was maintained for 6-8 weeks, after which it was treated with CO2 and oxalic acid dihydrate, and filtered under pressure. The residue was washed repeatedly to a syrup-like consistency, and filtrates combined; solvent evaporated under reduced pressure and the resultant product allowed to crystallize under refrigeration (Id.). The final product yield was still only about 10% (Id. at 485).
In an attempt to improve product yields, Lopez-Aparicio et al. reported the synthesis of 2-C-methyl-D-ribono-1,4-lactone from 2,3-O-isopropylidene-D-glyceraldehyde as an alternative to the Kiliani synthesis (Lopez-Aparicio et al., Carbohydrate Res., 129:99 (1984), as cited in F. J. Lopez-Herrera et al., J. Carbohydrate Chemistry, 13(5):767-775 (1994) at 768-769). The process of Lopez-Aparicio included condensing 2,3-O-isopropylidene-D-glyceraldehyde with (1-methoxy-carbonyl-ethylidene)triphenylphosphorane to produce methyl E-(S)-4,5-dihydroxy-4,5-O-isopropylidene-2-methyl-2-pentenoate; hydrolyzing (in HCl) and photochemically isomerizing the pentenoate; lactonizing the pentenoate product to produce a butenolide; tritylating the butenolide at C-5 by reaction with trityl-chloride and pyridine, followed by cis-hydroxylation with potassium permanganate and methylene chloride in the presence of a crown ether. Final removal of the trityl (triphenylmethyl) group was achieved by reaction with TFA (trifluoroacetic acid) (Id. at 768). Lopez-Aparicio et al. reported product yields of ribonolactone at about 80%, but others were not able to reproduce this figure based on the gram mass amounts of materials provided in the experimental section of their publication. Instead, calculations indicated a percent yield of about 36% ribonolactone. In addition, the process of Lopez-Aparicio et al. was far more complex than the Kiliani synthesis, required the use of toxic reagents such as potassium permanganate and specialized equipment for irradiation to attain photochemical isomerization, and had a minimum of 60 hours reaction time (Id. at 768, 770-772).
Walton et al. described the synthesis of 2′-C-methyladenosine from 2-C-methyl-D-ribono-lactone (Walton et al., J. Am. Chem. Soc., 88(19):4524-5 (1966)). In this case, the lactone was converted into its 2,3,5-tri-O-benzoyl derivative, and then reduced with bis(3-methyl-2-butyl)borane to provide an anomeric mixture of 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribofuranose (Id.). Attempts at separating the anomeric mixture both on acid-washed alumina and on silica gel resulted in rearrangement to 1,3,5-tetra-O-benzoyl-2-C-methyl-α-D-ribofuranose (Id.). In order to avoid rearrangement, the additional steps of treating the mixed anomers with benzoyl chloride in pyridine to obtain 1,2,3,5-tetra-O-benzoyl-2-C-methyl-(α)/(β)-D-ribofuranose, and of isolating the final product by chromatography were needed (Id.). Later Walton et al. described the synthesis of 2′-C-methyl-5-fluorocytidine, 2′-C-methyl-5-fluorouridine, and 2′- and 3′-C-methylcytidine via the Hilbert-Johnson reaction (Walton et al., Antiviral Nucleosides 12:306-309 (1969)). However, unexpectedly large amounts of O-glycoside formed when 2′-C-methylcytidine was synthesized from N-acetylcytosine-mercury, and mercury itself is a toxic reagent whose avoidance is desirable (Id.). In both synthetic procedures described by Walton et al., the final product yield was only about 11%.
In 1997 Harry-O'Kuru et al. described a synthetic route for preparing 2′-C-branched ribonucleosides (Harry-O'Kuru et al., J. Org. Chem., 62:1754-9 (1997)). Commercially available 1,3,5-tri-O-benzoyl-α-D-ribofuranose was used as the starting material, which was prepared from D-ribose or D-arabinose (D-arabinopyranose). The 1,3,5-tri-O-benzoyl-α-D-ribofuranose was oxidized at the free 2-OH with Dess-Martin periodinane reagent, and produced 1,3,5-tri-O-benzoyl-2-keto-ribofuranose as well as its corresponding hydrate. The desired product and hydrate were stirred with excess MgSO4 and permitted to stand overnight. The mixture was then filtered and concentrated in order to produce a substantially pure ketone product. The resultant 2-ketosugar was treated with MeMgBr/TiCl4 (or alternatively with MeTiCl3, CH2═CHMgBr/CeCl3, or TMSC≡CLi/CeCl3), which produced an anomeric mixture of the desired 1,3,5-tri-O-benzoyl-2-substituted alkyl-, alkenyl- or alkynyl-ribofuranoside and its transesterified isomers, α- and β-2,3,5-tri-O-benzoyl-2-substituted alkyl, alkenyl or alkynyl ribofuranoside in a nearly 5:3 ratio of desired product to isomeric forms (Id. at 1755). The 2-alkylated ribofuranosides then were converted to a single, desired product, 1,2,3,5-tetrabenzoyl-2-alkylribofuranoside, by treatment with benzoyl chloride, DMAP and triethylamine in approximately a 70% yield with a β/α ratio of 4:1 (Id.).
Beigelman et al. described the syntheses of 2′-C-methyl-nucleosides from D-glucose and D-ribose (Beigelman et al., Carbohydrate Research, 166:219-232 (1987)). Using D-glucose as a starting material, 1,2:5,6-di-O-isopropylidene-3-C-methyl-α-D-allofuranose was prepared and converted by selective incorporation of a p-methylbenzoyl group via a 5,6-O-dibutylstannylidene derivative (Id.). This was followed by treatment with aqueous 90% trifluoroacetic acid and periodate oxidation, elimination of the formyl group on the compound, and acetylation (Id.). Final product yield was about 77% (Id.). With D-ribose as a starting material, a 2,3-dimethyl-isopropylidene derivative with a protected 5-position was subjected to aldol condensation with formaldehyde, then treated with excess toluene-p-sulfonyl chloride in pyridine (Id.). The compounds were subsequently used to form a variety of products using conditions known in the art, including, for example, Kuhn methylation, reduction with LiAlH4 in THF, acid-catalyzed hydrolysis, and acetylation by boiling in excess Ac2O in pyridine (Id.). Average product yield was approximately 75-80%, but required costly materials and reagents (Id.).
Novak and Sorm detailed the preparation of crystalline 2-C-methyl-D-ribose and derivative compounds from 2-C-methyl-D-ribonolactone via sodium borohydride reduction (J. J. K. Novak & F. Sorm, Collection Czechoslov. Chem. Commun., 34: 857-866 (1969)). They characterized the nature of the hydroxyl group at the 2-position of the 2-C-methyl-ribofuranoside, particularly in comparison with the similarly situated hydroxyl group on the corresponding lactone (Id). While the hydroxy group on the lactone was easily acetylated under conditions known to those skilled in the art to afford 2,3,5-tri-O-acetyl and 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribonolactone, analogous conditions produced only 3,5-di-O-acetyl- and 3,5-di-O-benzoyl-2-C-methyl-D-ribofuranosides from 2-C-methyl-ribofuranosides (Id).
Later, Novak described chiro-optical properties of 2-C-methyl-1,4-lactones, which were prepared from D-lyxose and D-xylose via hypoiodite oxidation, and which had p-toluoyl protecting groups at C3 and C5 on the lactone (J. J. K. Novak, Collection Czechoslov. Chem. Commun., 39: 869-882 (1974)). In particular, a 2-CH3-ribono-1,4-lactone was synthesized by hydrolysis from 3,5-p-toluoyl-2-Br, 2-CH3-ribono-1,4-lactone (Id.). However, Novak described difficulty in separating protected lactone products from one another, and resulting syrup-like products when deblocking of the lactones by alkaline alcoholysis was attempted (Id. at 871).
Both Tokyo Tanabe Co., Ltd. (JP 61-212592) and BASF Aktiendgesellschaft (EP 0 288 847) reported epimerization processes for preparing unprotected D-ribose from D-arabinose, a common starting material for ribose production.
Tokyo Tanabe Co., Ltd., teaches the epimerization of aqueous D-arabinose in an organic solvent in the presence of a preferably molybdic (VI) acid and a boric acid compound, collection and passage of the reaction liquid through a 2- or 3-valent metal-type cation exchange material (a polystyrenesulfonic acid-type strongly acidic ion exchange resin converted to a Ca-type was preferred), elution with water to separate the unprotected ribose, and collection of the ribose compound (JP 61-212592, Abstract).
BASF teaches a continuous process in which an aqueous/alcoholic solution of D-arabinose is heated in a solvent in the presence of a basic anion exchanger loaded with a molybdenum (VI) compound. The eluate is collected and dried, methanol or ethanol added to the dried eluate and the mixture cooled to about 0° C. to crystallize unconverted D-arabinose which then is separated and recycled. The remaining filtrate is concentrated and purified according to methods known to those skilled in the art over a strongly acidic ion exchanger in the Ca2+ form, and any by product-free arabinose/ribose recycled into arabinose at the crystallization stage (EP 0288847).
Both the procedures of Tokyo Tanabe Co., Ltd. and BASF require sophisticated and expensive equipment and reagents, and the product compound has yet to have protecting groups added.
Japan Tobacco, Inc., prepared 3-DPA-lactone by protecting the 5-OH group on a gamma-ribonolactone, utilizing an acid chloride or acid anhydride with a tertiary amine to cause beta-elimination of the 3-OH and formation of a double bond between carbons 2 and 3 while simultaneously acylating the 2-OH group, and finally catalytically hydrogenating the double bond between C-2 and C-3 and removing the protective group to regenerate 5-OH. See EP 0 526,655 A1, EP 0 553,358 A1, and EP 0 553,358 B1, as well as their US equivalents U.S. Pat. No. 5,322,955 and U.S. Pat. No. 5,391,769.
Other related work on syntheses of ribonolactones and sugar analogues with protected substituents include the following.
Li et al., Organic Letters, 3(7):1025-28 (2001) synthesized 2′-C-β-trifluoromethyl pyrimidine ribonucleoside from 1,3,5-tri-O-benzoyl-α-D-ribofuranose, and then converted it to 3,5-di-O-benzoyl-2-C-β-trifluoromethyl-α-D-1-ribofuranosyl bromide. The latter bromide derivative compound was found to be an effective reaction intermediate in the formation of nucleosides.
Beigelman et al., Bioorg. Khim., 12(10):1359-65 (1986), synthesized 2-C-methyl-D-ribose derivative compounds via benzylation of 1,2:5,6-di-O-isopropylidene-3-C-methyl-α-D-allofuranose to form a first intermediate; hydrolyzed and selectively acylated the first intermediate to form 3-O-benzyl-1,2-O-isopropylidene-3-C-methyl-6-O-toluoyl-α-D-allofuranose; and sequentially deisopropylidenated, oxidized (with periodic acid), deformylated, acetylated, debenzylated and acetylated again to provide 1,2,3-tri-O-acetyl-2-C-methyl-5-O-toluoyl-β-D-ribofuranose as a final product.
Feast et al., Acta Chemica Scandinavica 19:1127-34 (1965), reported the preparation of α-D-glucosaccharinic acid, shown to be 2-C-methyl-D-ribo-pentonic acid, by alkaline treatment of D-fructose or 1-O-substituted D-fructose via a 1,4-lactone intermediate.
Kohn et al., J. Am. Chem. Soc., 87(23):5475-80 (1965), described a short route for obtaining a furanose derivative of an aldose, by reducing a tetraacyclohexono-gamma-lactone to its corresponding tetraacylhexofuranose through use of disiamylborane as a reducing agent. The reaction is particularly important for the formation of intermediates in the synthesis of C-1′ furanosyl nucleosides.
Kempe et al., Nucleic Acids Res., 10(21):6695-6714 (1982) reported the selective 2′-benzoylation at the cis 2′,3′-diols of protected ribonucleosides and isomerization of 2′-benzoates to 3′-benzoates. These protected nucleosides were used to synthesize oligoribonucleotides on solid silica gel supports, and subsequent deprotection resulted in the advantage of minimal internal nucleotide cleavage.
U.S. Pat. No. 4,294,766 to Schmidt et al. detailed the synthesis of pure ribonolactone from a mixture of ribonolactone and arabonolactone. Ribonolactone is an intermediate in the formation of riboflavin (vitamin B2). A mixture of potassium arabonate and potassium ribonate was “lactonized”, and the resulting lactone mixture, of which about 70% was ribonolactone, was separated by fractional crystallization using dioxane or ethylene glycol monomethyl ether. Lactonization was performed by methods known in the art, such as, for example, by using ion exchangers, or by concentrating the lactone in the presence of H2SO4 or K2SO4 and filtering off the precipitate.
Nucleoside Coupling
Walton described the synthesis of branched-chain nucleosides prepared by reacting 2,3,5-tri-O-acyl-2-(or 3)-C-alkylribofuranosyl halides with chloromercuric purine or pyrimidine compounds (U.S. Pat. No. 3,480,613). 3-Lower alkyl-D-ribofuranosyl halide intermediates were prepared starting from 1,2-O-isopropylidene-5-O-acyl-α-D-erythro-pentofuran-3-ulose by reacting this compound with a Grignard reagent to add a lower alkyl group at C3. Next, one of two pathways was followed: in the first pathway, the 5-O-acyl-1,2-O-isopropylidene-3-lower alkyl-D-ribofuranose was subjected to acidic alcoholysis to form an alkyl 5-O-acyl-3-lower-alkyl D-ribofuranoside; the latter compound was then acylated to the alkyl 2,3,5-tri-O-acyl-3-lower alkyl-D-ribofuranoside; and the resulting ribofuranoside could then be converted to a free sugar by subjecting it to a basic solvolysis and further hydrolysis in strong acid in aqueous medium, or converted to a halogenose by a halogen replacement reaction in appropriate solvent. In the second pathway, the 5-O-acyl-1,2-O-isopropylidene-3-lower alkyl-D-ribofuranose was acylated under basic conditions (pyridine) in inert solvent to form 3,5-di-O-acyl-1,2-O-isopropylidene-3-lower alkyl-D-ribofuranose, which was then hydrolyzed in strong acid and further acylated to provide the desired intermediates. 2-substituted, 6-substituted or 2,6-disubstituted purine nucleosides having a branched-chain at the 2′-position or 3′-position on the sugar moiety were then prepared by reacting 2,3,5-tri-O-acyl-D-ribofuranosyl halide with a chloromercuric 2,6-disubstituted purine at temperatures of 100° C. to 140° C. in a solvent such as toluene or xylene. Nucleosides having a desired pyrimidinone base were derived from 2,3,5-tri-O-acyl-2 (or 3)-C-lower alkyl-D-ribofuranosyl halide by reaction with a 2,4-dialkoxy-pyrimidine to form 1-(2,3,5-tri-O-acyl-2 (or 3)-C-lower alkyl-D-ribofuranosyl)-4-alkoxy-2(1H)-pyrimidone, which was then reacted with ammonia, or a primary or secondary amine to afford compounds having an amino substituent at the C-4 on pyrimidinone, or hydrolyzed under acidic or basic conditions to afford a pyrimidinone base having a hydroxy group at the C-4. Unfortunately, Walton's syntheses involve multiple steps, special conditions, and numerous, toxic reagents.
As shown in FIG. 5 the prior art teaches the coupling of 1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (4) with N4-benzoylcytosine using BSA in acetonitrile. The reaction mixture was heated to reflux for approximately 30 minutes, after which the Lewis acid, SnCl4, was added and the solution again heated to reflux for about 3.5 hours, to provide 4-NH-benzoyl-2′, 3′, 5′-tri-O-benzoyl-β-D-2′-C-methyl-cytidine (5a). Compound (5a) was obtained by dilution with ethyl acetate and aqueous, saturated sodium bicarbonate, and extensive chromatographic purification. Removal of the benzoyl protective groups was accomplished by overnight treatment of (5a) with a solution of methanol presaturated with ammonia to provide β-D-2′-C-methyl-cytidine (6).
Prodrugs
Pharmaceutically active compounds are sometimes administered in an esterified prodrug form. Carboxylic acid esters are used most commonly, while phosphonate and phosphate esters are used less frequently because they fail to hydrolyze in vivo and may produce toxic byproducts (see U.S. Pat. No. 6,312,662 to Erion et al.). Acyloxyalkyl esters are sometimes used as prodrugs for phosphate and phosphonate compounds, as are cyclic phosphonate esters and aryl esters, especially phenyl and benzyl esters (Farquhar et al., J. Pharm. Sci., (1983), 72(3):324; U.S. Pat. No. 6,312,662 to Erion et al.). Like nucleosides, phosphonic acids such as, for example, phosphonoformic acid and PMEA (Adefovir; 9-(2-phosphonylmethoxy-ethyl)adenine) show antiviral activity as do carboxylic acid or ether lipid prodrugs of nucleosides (U.S. Pat. No. 6,458,773 to Gosselin et al.).
Historically, prodrug syntheses and formulations have typically involved the 5′-position of a nucleoside or nucleoside analogue. Gosselin et al., supra, reported nucleosides in which the H of the 5′-OH group is replaced by any of the following: an acyl group including those in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic C1-C20 alkyl, phenyl or benzyl; a naturally-occurring or non-naturally-occurring amino acid; a 5′-ether lipid or a 5′-phosphoether lipid; alkoxyalkyl including methoxymethyl; aralkyl including benzyl; aryloxyalkyl such as, for example, phenoxymethyl; aryl including phenyl, optionally substituted with halogen, C1-C4 alkyl or C1-C4 alkoxy; a dicarboxylic acid such as, for example, succinic acid; a sulfonate ester such as, for example, an alkyl or aralkyl sulphonyl including methanesulfonyl; or a mono-, di-, or triphosphate ester.
Matulic-Adamic et al. (U.S. Pat. No. 6,248,878) reported the synthesis of nucleoside analogues that comprise a ribofuranose ring with a phosphorus-containing group attached to the 3′-position via an oxygen atom and a substituted pyrimidine base. The phosphorus-containing group includes dithioates or phosphoramidites, or may be part of an oligonucleotide. These compounds are prodrugs because they are reacted further to provide final, desired nucleosides and nucleoside analogues. The compounds are synthesized in a multi-step process that couples, as starting materials, a ribofuranose having an hydroxy or acetoxy group at C-1 and benzoyl-protecting groups at C-2-, C-3 and C-5, and a 4-OSiMe3 pyrimidine to produce an 1-(2,3,5-tri-O-benzoyl-ribo-furanosyl)-pyrimidin-4-one; then adds ammonia in methanol to the product of the first reaction in order to remove the benzoyl protecting groups; then reacts DMT-Cl/Pyr reacted with the unprotected product compound, which results in the addition of DMT to the 5′-O position of ribofuranose; then reacts TBDMS-Cl, AgNO3, and Pyr/THF with the 5′-O-DMT substituted ribofuranose; and finally performs standard phosphitylation to produce the phosphorus-containing group located at the 3′-O. Each of the syntheses presented include at least 4 to 7 steps.
Chu et al. described prodrugs that are azide derivative compounds and compositions, including nucleoside and phosphorylated nucleoside analogues (U.S. Pat. No. 6,271,212). Such azide prodrugs have as advantages their ability to cross the blood-brain barrier, provide a longer half-life, and afford greater bioavailability and increased stability of the active form of the drug than previously observed. However, Chu et al. reported a lengthy, multi-step synthesis required for preparing their azide prodrugs.
Borretzen et al. described antiviral prodrugs that were nucleosides and nucleoside analogues. They reported certain fatty acid esters of anti-viral nucleosides and nucleoside analogues where the fatty acid in a mono-unsaturated C18 or C20 fatty acid was bonded to the 5′-position of the nucleoside or nucleoside analogue through an acylation process (U.S. Pat. No. 6,153,594). The process was carried out in the presence of a catalyst, and was allowed to proceed for 24-60 hours. Product isolation was accomplished by extraction with an organic solvent, and purification by chromatography and/or recrystallization from an appropriate solvent. Percent yield of the product varied widely from 15-82%. Borretzen et al., however, did not use the term “prodrug”.
In 1999, McCormick et al. described the carbonate formation at the 3′-OH of guanosine, using an unprotected ribose as a starting material (McCormick et al., J. Am. Chem. Soc. 1999, 121(24):5661-5). McCormick was able to synthesize the compound by a sequential, stepwise introduction of the O- and N-glycosidic linkages, application of certain protecting groups, sulfonation and final deprotection. As one step in their process, McCormick et al. reacted unprotected guanosine with BOC-anhydride, DMAP, Et3N, and DMSO at room temperature for 4 hours to obtain directly a carbonate at the 3′-OH of guanosine.
Also in 1999, Tang et al. disclosed a process for preparing phosphoramidite prodrugs of 2′-C-β-methyl-cytidine ribonucleosides (Tang et al., J. Org. Chem., 1999, 64:747-754). Like many of their colleagues, Tang et al. reacted 1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D ribofuranose with persilylated 4-N-benzoylcytosine in the presence of the Lewis acid, SnCl4, as a first step in their synthesis (Id. at 748, Scheme 1a).
In 2000, Novirio Pharmaceuticals (now Idenix) discovered that the stability and bioavailability of antiviral nucleoside analogues is enhanced by the administration of amino acid ester forms of antiviral nucleosides (U.S. Ser. No. 09/864,078, pending; U.S. Ser. No. 10/261,327, pending; WO 01/90121; and U.S. Provisional Application Nos. 60/377,983 and 60/392,351). Processes used for preparing these amino acid esters of nucleosides and nucleoside analogues began with appropriately branched β-D or β-L nucleosides that optionally could be protected by an appropriate protecting group such as, for example, a silyl group, and subsequently deprotected, by methods known to those skilled in the art (Zhang et al., Tetrahedron Letters, 1992, 33:1177-80; Greene et al., Protective Groups in Organic Synthesis, John Wiley & Sons, 2nd Edition (1991); Kerr et al., J. Pharmaceutical Sciences, 1994, 83:582-6; Tang et al., J. Org. Chem., 1999, 64(3): 747-754; and Cavelier et al., Tetrahedron Letters, 1996, 37:5131-4). The optionally protected branched nucleoside was then coupled with a suitable acyl donor, such as an acyl chloride and/or an acyl anhydride or an activated acid, in an appropriate protic or aprotic solvent and at a suitable reaction temperature, to provide the 2′ or 3′ prodrug of a 1′, 2′, 3′ or 4′ branched β-D or β-L nucleoside, optionally in the presence of a suitable coupling agent (see Synthetic Communications, 1978, 8(5): 327-33; J. Am. Chem. Soc., 1999, 121(24):5661-5; Bryant et al., Antimicrob. Agents Chemother., 2001, 45, 229-235; Standring et al., Antiviral Chem. & Chemother., 2001, 12 (Suppl. 1), 119-129; Benzaria et al., Antiviral Res., 2001, 50, A79; Pierra et al., Antiviral Res., 2001, 50, A79; and Cretton-Scott et al., Antiviral Res., 2001, 50, A44). Possible coupling reagents are any reagents that enable compounds or moieties to be linked to one another including, but not limited to, various carbodiimides, CDI, BOP and carbonyldiimidazole. For example, for a 3′-prodrug of a 2′-branched nucleoside, the nucleoside preferably was not protected, but was coupled directly to an alkanoic or amino acid residue via a carbodiimide-coupling reagent.
The prior art process shown in FIG. 5 included the following reaction sequence for preparing a 3′-valinyl ester nucleoside prodrug of cytidine: 1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (4) was added to a mixture of BSA and N4-benzoylcytosine in acetonitrile and heated to reflux for approximately 30 minutes, after which the Lewis acid, 5 nCl4, was added and the solution again heated to reflux for about 3.5 hours, to provide 4-NH-benzoyl-2′, 3′, 5′-tri-O-benzoyl-β-D-2′-C-methyl-cytidine (5a). Compound (5a) was obtained by dilution with ethyl acetate and aqueous, saturated sodium bicarbonate, and extensive chromatographic purification. Removal of the benzoyl protective groups was accomplished by overnight treatment of (5a) with a solution of methanol presaturated with ammonia to provide β-D-2′-C-methyl-cytidine (6). Compound (6) in DMF was reacted with N,N-dimethylformamide dimethyl acetal at room temperature for approximately 1.5 hours, to provide cytidine having a protected amino group at C4, N4-[(dimethylamino)methylene]-β-D-2′-C-methyl-cytidine (2); a solution of amino-protected cytidine (7) in dry pyridine next was reacted with imidazole and TBDPSCl at room temperature for approximately 6 hours to afford cytidine whose 5′-O was silyl-protected (8); N—Boc-L-Valine in the presence of DEC, DMAP, and THF/DMF then were added to the 4- and 5′-protected, β-D-2′-C-methyl-cytidine (8) at room temperature for approximately 2 days to produce a 4- and 5′-protected, 3′-O—L—N—BOC-valinyl ester of β-D-2′-C-methyl-cytidine (2); the 4- and 5′-protected, 3′-O—L—N—BOC-valinyl ester of β-D 2′-C-methyl-cytidine (2) was taken up in dry methanol to which was added ammonium fluoride and the mixture brought to reflux in order to remove the 5′-silyl and 4-amino protecting groups, producing 3′-O—L—N-(tert-butoxycarbonyl) valinyl ester of β-D-2′-C-methyl cytidine (10), which was purified by column chromatography; and finally, to a solution of 3′-O—L—N-(tert-butoxycarbonyl) valinyl ester of β-D-2′-C-methyl cytidine (10) in dry ethyl acetate was added a 20% solution of HCl/ethyl acetate and the mixture stirred for about 2 hours to remove the BOC-protecting group, thereby providing the hydrochloride salt of 3′-O-valinyl ester of β-D-2′-C-methyl-cytidine as a final product (11). The prior art synthesis shown in FIG. 6 used uracil in place of the benzoyl cytosine to prepare compound (11), β-D-2′-C-methyl-cytidine.
In view of the above, it would be advantageous to have an efficient process for preparing a nucleoside or a nucleoside analog, such as a 2′-methyl-nucleoside or a 2′-methyl-3′-O-valinyl-nucleoside, their intermediates, including the 2-C-methyl-ribonolactone and 2-C-methyl-D-ribofuranose, and their salts and/or prodrugs thereof.
It is another object of the present invention to provide a process for the selective addition of a group at the 3′-OH of a nucleoside that would render the derivative compound a prodrug.
It is yet another object of the present invention to have an efficient process for preparing protected sugar analogue compounds that involves a minimal number of steps, and utilizes an inexpensive starting material.
It is yet another object of the present invention to decrease significantly the time required for preparing protected sugar intermediates as compared to other processes for synthesizing similar products.
Further, it is another object of the invention to have a process that runs to completion in a matter of hours and provides a final product high in both yield and purity.
It is yet another object of the invention to have a process that employs easy-to-use, non-toxic reagents, and whose final product is easily isolated by techniques commonly known in the art and easily scaleable.
It is still another object of the present invention to obtain the final product compound in high yields and purity exceeding at least 90 or 95%.
It is a further object of the present invention to employ non-toxic, easily handleable reagents.